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Objectives: A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production.Materials and methods: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of -lactamases. PCR assays with primers specific for the blaVIM gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo--lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the blaVIM-1 gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the blaVIM-1 gene as a probe, amplified by PCR.Results: The isolate was resistant to extended-spectrum -lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 108?cfu/mL was used. Isoelectric focusing detected a -lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the blaVIM-1 gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The blaVIM-1 probe hybridized with an approximately 130?kb fragment of genomic DNA, suggesting a chromosomal location of the gene.Conclusion: We describe a novel class 1 integron containing blaVIM-1 and aac(6')-IIc genes in an E. cloacae clinical isolate. [ABSTRACT FROM AUTHOR]

: Objectives Clinical isolates of Klebsiella pneumoniae (91), Escherichia coli (49), Enterobacter spp. (27), Proteus mirabilis (17), Citrobacter freundii (2), Providencia stuartii (3) and Serratia spp. (5), with various MICs of imipenem, were examined for production of metallo-β-lactamases (MBLs) with different phenotypic laboratory tests that have been previously published to detect MBLs in Pseudomonas aeruginosa and Acinetobacter spp. : Methods A total of 194 (95 MBL-positive and 99 MBL-negative) clinical isolates with imipenem MICs ≤0.25 to >256 mg/L were examined. All isolates were evaluated by the double-disc synergy test (DDST), the combination disc test (CDT), the MBL Etest and the modified Hodge test. The presence of blaVIM and blaIMP genes was evaluated by in situ hybridization with specific probes and was certified by PCR. : Results In 30 blaVIM-positive isolates that exhibited MICs of imipenem ≤4 mg/L, MBL Etest could not be evaluated. CDT with ceftazidime and 1900 or 750 µg of EDTA, and DDST after applying an imipenem disc 10 mm apart from a disc containing ∼1900 µg of EDTA, showed the highest sensitivity (97.9% to 100%) and specificity (87.9% to 96%) rates among the analysed procedures. CDT with imipenem and 1900 µg of EDTA exhibited a sensitivity of 94.7% and showed very good specificity (98%). : Conclusions The CDT with imipenem/imipenem+0.5 M EDTA or ceftazidime/ceftazidime+0.2 M EDTA and the DDST with imipenem 10 mm apart from EDTA are the most effective methods for the detection of MBLs in Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Objectives Infections due to multidrug-resistant (MDR) Gram-negative pathogens in the ICU have prompted the use of colistin, an antibiotic forgotten for decades. The aim of this retrospective observational study was to record and present the emergence of colistin-resistant Klebsiella pneumoniae (CRKB) in a Greek ICU. Methods In a new university tertiary hospital, the first patients admitted in the ICU were already colonized or infected with MDR pathogens, and this led to frequent colistin use as part of empirical or microbiologically documented therapy. Colistin resistance was defined as MIC >4 mg/L by the Etest method. All CRKB isolated in surveillance cultures or clinical specimens in the ICU during the period 2004–5 were recorded along with patients' characteristics. Results Eighteen CRKB were isolated from 13 patients over a 16 month period, representing either colonizing or infective isolates. Patients' mean age was 70 years, with a mean APACHE II score at admission of 22. They all had a long hospitalization (median 69 days) and a long administration of colistin (median 27 days). Colistin-resistant isolates were implicated as pathogens in two bacteraemias, a ventilator-associated pneumonia and two soft tissue infections. Repetitive extragenic palindromic PCR identified six distinct clones, and horizontal transmission was also documented. Conclusions Selective pressure due to extensive or inadequate colistin use may lead to the emergence of colistin resistance among K. pneumoniae isolates, jeopardizing treatment options in the ICU, potentially increasing morbidity and mortality of critically ill patients and necessitating prudent use of colistin. [ABSTRACT FROM AUTHOR]